One of the major objectives in chromatin research is to ascertain how the higher order organization of chromatin (i.e., that beyond the nucleosome per se) influences nuclear functions such as transcription, replication and cell-cycle control. This currently cannot be accomplished because there is no technical means to rigorously characterize the higher order structural features of in vivo-assembled chromatin. In this R21 "Exploratory Studies for HighRisk/High Impact Research" proposal, this limiting technical barrier will be overcome by developing quantitative agarose gel electrophoresis as a means to characterize the extent of higher order folding, flexibility, and surface charge density of two different well defined in vivo- assembled chromatin systems. In Specific Aim 1, the quantitative electrophoretic approach will be applied to the analysis of the higher order organization of yeast minichromosomes that have been assembled in vivo from both native core histones as well as core histones expressing a variety of mutations in their biologically important N-terminal domains. In Specific Aim 2, the effect of chromatin higher order organization on transcriptional regulation will be examined. This will be accomplished by first determining the higher order folding, flexibility, and surface charge density of the mouse mammary tumor virus long-terminal repeat (MMTV-LTR) promoter before and after steroid hormone activation of MMTV-LTR transcription. Also in Specific Aim 2, the higher order folding, flexibility, and surface charge density of the transcriptionally active TALS minichromosome isolated from yeast a cells will be compared with the structural properties of the same minichromosome isolated from alpha cells, where it is transcriptionally repressed. The proposed studies will be the first experiments of any type to directly characterize the higher order structural properties of in vivo-assembled chromatin, and in doing so will overcome a major barrier facing chromatin research. It subsequently will become possible to study the structural properties of numerous functionally important in vivo- assembled chromatin regions (e.g., promoters, enhancers), and other functionally important in vivo-assembled multi-component complexes as well.